This application is based upon the conviction that activation of factor IX (FIX) by the tissue factor/factor VIIa (TF/VIIa) complex represents a key step in the initiation of coagulation by the extrinsic pathway. All of the specific aims represent continuation of our ongoing studies. FIX consists of an N-terminal Gla domain followed by two EGFlike domains, an activation peptide (AP) segment, and a C-terminal serine protease domain. In specific aim 1, we will address the hypothesis that the protease and EGF2 domains of FIXa play a primary role in binding to factor VIIIa (FVIIIa). A select number of FIXa mutants in these two domains will be investigated for their abilities to interact with FVIIIa in both kinetic and direct binding assays. In the kinetic based tenase assays, we will use FVIIIa and its isolated A2 subunit. In direct binding assays, we will use the isolated A1, A2, A3-C1, and A3-C1-C2 subunits of FVIIIa. In specific aim 2, we will address the hypothesis that the Gla and EGFI domains as well as the AP segment of FIX play a key role in its interaction with TF/VIIa. Here, we will replace the Gla domain of FIX with that of protein C as well as make point mutants in the EGF1 domain. The ability of these FIX mutants to bind to soluble TF (sTF) and to be activated by TF/VIIa (and sTF/VIIa) or FXIa will be determinedx We believe that, in contrast to the mutants in specific aim 1, some of the EGF1 and Gla domain mutants will be impaired in binding to sTF as well as in activation by TF/VIIa (and sTF/VIIa). Further, the EGF 1 and Gla domain mutants may be impaired to a different degree in binding to FVIIIa. The binding mode of the AP segment of FIX will be studied by displacement of benzamidine from our benzamidine-VIIa/sTF crystals by the synthetic AP segments and structure determinations of the resultant AP-sTF/VIIa complexes. In specific aim 3, we hypothesize that there is a sodium site in the protease domain of FIXa, which is similar to FXa, FVIIa, and activated protein C but not to thrombin. We will investigate the effect of sodium on the catalytic efficiency of FIXa and on binding to FVIIIa as well as evaluate a functional linkage between the S 1 site, sodium site, and the calcium site in the protease domain of FIXa. To experimentally define sodium site, we will prepare EGF2-protease segment of FIXa and crystallize it in the presence of (a) sodium and (b) rubidium and determine its structure. Our integrated approach is expected to provide new information regarding the activation of FIX by TF/VIIa and the assembly of FIXa:FVIIIa complex, which could be central to developing a new generation of antithrombotics.